Cytokines and chemokines represent two important groups of proteins that control the immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation leading to various diseases, including rheumatoid arthritis, characterized by chronic inflammation and bone erosion. Chemokine activity is regulated at multiple levels, such as post-translational modification (PTM) of chemokines and their receptors by specific enzymes including proteases and peptidylarginine deiminases. Many in vitro experiments underscore the importance of post-translational processing of human chemokines. PTMs may enhance or reduce chemokine activity or may alter the receptor specificity of chemokine ligands. However, identification of chemokine isoforms in physiological in vivo settings forms the ultimate proof that PTM of chemokines is relevant in regulating the biological activity of these molecules. This review summarizes current knowledge on the in vivo role for PTMs in the regulation of chemokine activity.